Influence of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus

Abstract

The growth phase-dependent activity profile of the alternate transcription factor sigma(B) and its effects on the expression of sar and agr were examined in three different Staphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant sigma(B) activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective in rsbU. sigma(B) activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3 (sarP1-P2-P3)-driven firefly luciferase (luc+) reporter gene fusion demonstrated a strong sigma(B) activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or Delta rsbUVWsigB mutants. In contrast, expression of the agr locus, as measured by RNAIII levels and by an hldp::luc+ fusion, was found to be higher in the absence of sigma(B) activity, such as in rsbU or Delta rsbUVWsigB mutants, than in wild-type strains. Overexpression of sigma(B) in BB255 derivatives resulted in a clear increase in sarP1-P2-P3::luc+ expression as well as a strong decrease in hldp::luc+ expression. The data presented here suggest that sigma(B) increases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.

FIG. 1

Genetic organization of the sar and agr loci of S. aureus. Genetic organization of the sar locus (A) and the agr locus (B) of S. aureus and schematic representation of the integration of sarP1-P2-P3::luc+ or hldp::luc+ fusion constructs into the S. aureus chromosome by single crossover. For a description of construction of the plasmids pECsarP1-P2-P3-luc+ and pEChldp-luc+ and integration of the constructs into the S. aureus chromosome, see Materials and Methods. Open reading frames, promoters, and respective transcripts are indicated.

FIG. 2

ς^B activity during growth of S. aureus. Expression of asp23::luc+ during growth of S. aureus strains BB255 (A), MSSA1112 (B), Newman (C), and their respective sigB mutants. Strains were grown in LB medium at 37°C. Bacterial growth was measured as the OD₆₀₀ (solid symbols). ς^B transcriptional activity was determined by measuring the luciferase activity of Luc+ (open symbols), the product of the luc+ reporter gene fused to the ς^B-dependent promoters of asp23 (asp23p). (A) Squares, S. aureus strain MB33 (BB255, asp23p::luc+); triangles, strain MB49 (BB255, rsbU+ asp23p::luc+); circles, strain MB90 (BB255, ΔrsbUVWsigB asp23p::luc+). (B) Squares, S. aureus strain MB73 (MSSA1112, asp23p::luc+); circles, strain MB70 (MSSA1112, ΔrsbUVWsigB asp23p::luc+). (C) Squares, S. aureus strain MB32 (Newman, asp23p::luc+); circles, strain MB69 (Newman, ΔrsbUVWsigB asp23p::luc+).

FIG. 3

Northern blot analyses of the sar locus. Total RNAs (8 μg/lane) of S. aureus strains BB255 (A), MSSA1112 (B), Newman (C), and their respective sigB mutants were blotted onto positively charged nylon membranes and subjected to Northern blot analyses. RNAs were obtained from cells grown in LB medium at 37°C and harvested at different growth stages (indicated as OD₆₀₀ values [numbers above the lanes]). The blotted membranes were hybridized using a digoxigenin-labeled DNA probe specific for sarA (for details on the construction, see Materials and Methods). The RNA molecular weight marker I (Roche) was used as a size marker. Relevant transcript signals are indicated.

FIG. 4

Role of ς^B in the regulation of sarP1-P2-P3::luc+ expression during growth. S. aureus derivatives of strains BB255 (A), MSSA 1112 (B), Newman (C), and their respective sigB mutants were grown in LB medium at 37°C. Bacterial growth was measured by OD₆₀₀ (closed symbols). sarP1-P2-P3::luc+ expression was determined by measuring the luciferase activity of the reporter gene luc+ (open symbols). (A) Squares, S. aureus strain MB98 (BB255, sarP1-P2-P3::luc+); circles, strain MB102 (BB255, sigB sarP1-P2-P3::luc+). (B) Squares, S. aureus strain MB105 (MSSA1112, sarP1-P2-P3::luc+); circles, strain MB113 (MSSA1112, sigB sarP1-P2-P3::luc+). (C) Squares, S. aureus strain MB100 (Newman, sarP1-P2-P3::luc+); circles, strain MB101 (Newman, sigB sarP1-P2-P3::luc+).

FIG. 5

Effect of overexpressed ς^B on the expression of sarP1-P2-P3::luc+. S. aureus derivatives of strain MB98 (BB255, sarP1-P2-P3::luc+), harboring plasmid pIK64 (P_xyl::sigB) (A) or control plasmid pTX15 (P_xyl) (B) were grown in LB medium at 37°C. Growth was measured as the OD₆₀₀ (closed symbols). Growing cultures were split into equal parts at the OD₆₀₀ of 1, and overexpression of ς^B was induced in one of the parts by supplementing 0.5% xylose (diamonds), while the control part was left without addition (squares). The arrow denotes the time point of supplementation. sarP1-P2-P3::luc+ expression (open symbols) was determined by measuring the luciferase activity of the reporter gene luc+ as described in Materials and Methods.

FIG. 6

Northern blot analyses of RNAIII. Total RNAs (8 μg/lane) of S. aureus strains BB255 and GP268 (BB255, rsbU⁺) (A), MSSA1112 and MB39 (MSSA1112, ΔrsbUVWsigB) (B), and Newman and IK 184 (Newman, ΔrsbUVWsigB) (C) were blotted onto positively charged nylon membranes and subjected to Northern blot analyses. RNAs were obtained from cells grown in LB medium at 37°C and harvested at different growth stages (indicated as OD₆₀₀ values above the lanes). The blotted membranes were hybridized using a digoxigenin-labeled DNA probe specific for RNAIII (for details on construction, see Materials and Methods). The RNA molecular weight marker I (Roche) was used as the size marker. Relevant transcript signals are indicated.

FIG. 7

Role of ς^B in the regulation of hldP::luc+ expression during growth. S. aureus derivatives of strains BB255 (A), MSSA1112 (B), and Newman (C) were grown in LB medium at 37°C. Bacterial growth was measured by OD₆₀₀ (closed symbols). hldp::luc+ expression was determined by measuring the luciferase activity of the reporter gene luc+ (open symbols) as described in Materials and Methods. (A) Squares, S. aureus strain MB95 (BB255, hldp::luc+); diamonds, strain MB94 (BB255, rsbU⁺ hldp::luc+). (B) Squares, S. aureus strain MB104 (MSSA1112, hldp::luc+); circles, strain MB112 (MSSA1112, sigB hldp::luc+). (C) Squares, S. aureus strain MB97 (Newman, hldp::luc+); circles, strain MB103 (Newman, sigB hldp::luc+).

FIG. 8

Effect of overexpressed ς^B on the expression of hldP::luc+. S. aureus derivatives of strain MB95 (BB255 hldp::luc+), harboring plasmid pIK64 (P_xyl::sigB) (A) or control plasmid pTX15 (P_xyl) (B), were grown in LB medium at 37°C. Growth was measured as the OD₆₀₀ (closed symbols). Growing cultures were split into equal parts at the OD₆₀₀ of 1, and overexpression of ς^B was induced in one of the parts by supplementing 0.5% xylose (diamonds), while the control part was left without addition (squares). The arrow denotes the time point of supplementation. hldp::luc+ expression (open symbols) was determined by measuring the luciferase activity of the reporter gene luc+ as described in Materials and Methods.